Schain L R, Jain S, Wysocki M, Hall M, Dadey B, Pennathur-Das R, Biddle W, Wolf J, Okarma T B, Lebkowski J S
Applied Immune Sciences, Inc., Santa Clara, CA 95054, USA.
J Hematother. 1997 Aug;6(4):335-49. doi: 10.1089/scd.1.1997.6.335.
The isolation and culture of human CD34+ cells could have broad clinical application for hematologic support following high-dose chemotherapy or bone marrow transplantation. The need for reproducible, animal product-free conditions for the culture of progenitors is crucial to the widespread clinical implementation of ex vivo cell therapies. In these studies, we explored the use of animal serum-free (ASF) medium for the culture of isolated human bone marrow and peripheral blood CD34+ cells. In this ASF system, isolated CD34+ cells were cultured using a variety of different growth factor combinations. Such ASF culture conditions yielded equivalent to superior cell and progenitor growth when directly compared with culture containing 10% fetal calf serum (FCS). In cultures containing IL-1, IL-3, and stem cell factor, total cell numbers increased, on average, 33-fold over the first 2 weeks. On phenotypic analysis, the ASF cultures demonstrated sustained proliferation of CD33+ myeloid cells throughout the culture period. CD34+ cell numbers increased during the first 7-10 days of culture, with a mean 3.4-fold expansion. Concomitant with the CD34+ cell expansion was an average 8.2-fold expansion of colony-forming unit-granulocyte-macrophage (CFU-GM) and a 102.0-fold increase in burst-forming units-erythrocytes (BFU-E). Likewise, a mean 4929-fold expansion of CD41a+ megakaryocyte progenitors was observed in these CD34+ cultures. Different combinations of growth factors affected the fold increase in cell and progenitor number. When CD34+ cell cultures from normal healthy volunteers mobilized with either G-CSF or GM-CSF were compared, similar expansions of total cell and progenitor cells resulted. However, CD41+ cells expansions were greater in those samples from G-CSF-mobilized volunteers in every case tested. These studies established the feasibility of this ASF CD34+ cell culture system to generate a population of maturing progenitors for potential use in transfusion support during cytopenic periods following high-dose chemotherapy or bone marrow transplantation.
人CD34+细胞的分离和培养在大剂量化疗或骨髓移植后的血液学支持方面可能具有广泛的临床应用。对于祖细胞培养而言，需要可重复的、无动物产品的条件对于体外细胞治疗在临床上的广泛应用至关重要。在这些研究中，我们探索了使用无动物血清（ASF）培养基来培养分离出的人骨髓和外周血CD34+细胞。在这个ASF系统中，使用多种不同的生长因子组合来培养分离出的CD34+细胞。与含有10%胎牛血清（FCS）的培养物直接比较时，这种ASF培养条件产生的细胞和祖细胞生长相当或更优。在含有白细胞介素-1、白细胞介素-3和干细胞因子的培养物中，在前两周内总细胞数平均增加了33倍。通过表型分析，ASF培养物在整个培养期间显示出CD33+髓样细胞的持续增殖。CD34+细胞数量在培养的前7至10天增加，平均扩增3.4倍。与CD34+细胞扩增同时发生的是集落形成单位 - 粒细胞 - 巨噬细胞（CFU - GM）平均扩增8.2倍以及爆式红系集落形成单位（BFU - E）增加102.0倍。同样，在这些CD34+培养物中观察到CD41a+巨核细胞祖细胞平均扩增4929倍。不同的生长因子组合影响细胞和祖细胞数量的增加倍数。当比较用粒细胞集落刺激因子（G - CSF）或粒细胞 - 巨噬细胞集落刺激因子（GM - CSF）动员的正常健康志愿者的CD34+细胞培养物时，总细胞和祖细胞产生了相似的扩增。然而，在每种测试情况下，来自G - CSF动员志愿者的样本中CD41+细胞的扩增更大。这些研究确立了这种ASF CD34+细胞培养系统用于生成一群成熟祖细胞的可行性，这些祖细胞可用于在大剂量化疗或骨髓移植后的血细胞减少期提供输血支持。
